Eiji Ichimura

Expression of c-met/HGF Receptor in Human Non-small Cell Lung Carcinomas in vitro and in vivo and Its Prognostic Significance

The expression of c‐met/HGF receptor was evaluated in non‐small cell lung cancers (NSCLC) by western blot analysis of 11 established cell lines and 104 surgically resected tissues. All cancer cell lines (eight adenocarcinomas, two squamous cell carcinomas and a large cell carcinoma) showed strong c‐met protein bands of 145 kDa and 170 kDa. Moreover, c‐met protein was demonstrated in 34 (72.3%) of 47 surgically resected adenocarcinomas, 20 (38.5%) of 52 squamous cell carcinomas and 3 of 5 others, and the results were mostly confirmed immunohistochemically in formalin‐fixed and paraffin‐embedded tumors of the same case. Although squamous cell carcinomas showed relatively high c‐met protein expression in established cell lines, more adenocarcinomas than squamous cell carcinomas showed c‐met protein expression in the original cancers. Furthermore, two cell lines used in this study originated from primary cancers negative for c‐met protein expression, suggesting that c‐met protein expression might be influenced by cultivation. Furthermore, clinicopathological study revealed that NSCLC with c‐met protein expression tended to be in a higher pathological tumor stage and to have a worse outcome than those without such expression. In conclusion, c‐met protein is expressed in cell lines and primary tumors of NSCLC, and this phenomenon is probably closely related to the aggressive behavior or progression of NSCLC, especially of adenocarcinomas.

Coexpression of HGF and c-Met/HGF receptor in human bone and soft tissue tumors

To understand the interaction between hepatocyte growth factor (HGF) and its receptor c‐Met on various bone and soft tissue tumors, their expressions were investigated by western blot analysts, immunohistochemistry and enzyme immunoassay. Western blot analysis revealed that c‐Met protein was expressed in 21 (38.8%) of 54 tumors, which detailed to seven (25.9%) of 27 bone tumors and 14 (51.8%) of 27 soft tissue tumors. Most malignant fibrous histiocy‐tomas (MFH) and all neurofibromas expressed c‐Met protein. The highest expression of c‐Met protein was seen in a case of biphasic synovial sarcoma, where its immunoreac‐tivity was localized only on the epithelial component and not on the sarcomatous component. By enzyme immunoassay for HGF, all but one MFH showed HGF production and the mean level of HGF was the highest among the tumors investigated. Neurofibmmas and osteosarcomas had the next highest mean levels of HGF production, respectively. Coexpression of HGF and c‐Met was obsewed in 19 (35.2%) of 54 tumors and was frequently observed in neurofibroma, followed by MFH and synovial sarcoma. Although the mode of interaction between HGF and c‐Met varies among the various bone and soft tissue tumors including MFH, their signaling system may play an Important role in the development and progression of bone and soft tissue tumors.

Metabolism and hepatic toxicity of flutamide in cytochrome P450 1A2 knockout SV129 mice.

BackgroundFlutamide, a nonsteroidal antiandrogen used for treatment of prostate cancer, causes a temporary increase in transaminase and in some cases severe liver dysfunction. It is dominantly metabolized by cytochrome P450 (CYP) 1A2 into 2-hydroxyflutamide (OH-flutamide), which has stronger antiandrogenic activity without obvious cytotoxicity to cultured hepatocytes. We hypothesized that another subsidiary metabolite might be responsible for induction of hepatotoxicity.MethodsFlutamide was administered daily to CYP1A2 knockout mice and parental SV129 mice to compare pharmacokinetics and appearance of hepatic toxicity.ResultsIn the CYP1A2 knockout mice, the plasma concentration of flutamide maintained at a high level and OH-flutamide stayed low; a higher amount of FLU-1, an alternative metabolite of flutamide, was detected in urine. Simple repetitive administration of 800 mg/kg of flutamide for 28 days to CYP1A2 knockout mice did not show abnormal elevation of plasma alanine aminotransferase (ALT). However, after the knockout mice were fed with an amino acid-deficient diet for 2 weeks, which reduced the glutathione (GSH) content to 27% of the initial, administration of 400 mg/kg of flutamide increased ALT to over 200 IU/l and histopathologically moderate hepatitis developed. Since FLU-1 itself did not show cytotoxicity or reduce GSH content in vitro, a further metabolized molecule must cause the hepatotoxicity.ConclusionsBlockade of CYP1A2 produced an unknown potential hepatotoxic molecule through FLU-1, and GSH might play an important role in diminishing the reactive hepatotoxic metabolite.

Immunohistochemical expression of aminopeptidase N (CD13) in human lung squamous cell carcinomas, with special reference to Bestatin adjuvant therapy.

Bestatin, a specific inhibitor of aminopeptidase N (CD13), has been reported to prolong survival time in patients with completely resected stage I lung squamous cell carcinoma. Considering the antitumor mechanism of Bestatin, it is interesting to know whether CD13 is expressed in human lung squamous cell carcinoma. The immunohistochemical expression of CD13 was examined in human lung carcinoma and the question of whether CD13 was immunohistochemically expressed in the interstitial tissue was investigated, mainly in the fibroblasts and blood vessels, surrounding the tumor nests of various kinds of non‐small cell lung cancers, especially of squamous cell carcinomas. In Japanese squamous cell carcinoma of the lung, 38 (61.3%) out of 62 cancers were positively stained in the same manner on immunohistochemistry for CD13. The area of interstitial tissue positively stained for CD13 varied depending on the case. To confirm the cell nature of the interstitial tissue with CD13 positivity, double immunohistochemistry using CD34 and α‐smooth muscle actin was performed. Double immunohistochemistry showed that the majority of CD13‐positive cells were slender fibroblastic cells around the blood vessels and some endothelial cells.

Antimyeloma activity of NK012, a micelle‐forming macromolecular prodrug of SN‐38, in an orthotopic model

NK012 is a micelle‐forming macromolecular prodrug of 7‐ethyl‐10‐hydroxy camptothecin (SN‐38), an active metabolite of irinotecan. It is accumulated and retained in tumor tissues and gradually releases SN‐38 in an enzyme‐independent manner. NK012 was previously demonstrated to have stronger antitumor activity than irinotecan in a broad range of human solid‐tumor xenograft models. In our study, we used an orthotopic multiple myeloma (MM) model created by injecting CD138‐positive U266B1, a myeloma cell line that produces human IgE lambda light chain (monoclonal protein, M protein), into immunodeficient NOD/Shi‐scid, IL‐2Rγcnull mice. This model shows typical bone marrow infiltration by the human myeloma cells. We evaluated the antimyeloma activity of intravenously administered NK012 in this model and showed that it suppressed the M protein concentration in the plasma and proliferation of myeloma cells in the bone marrow in a dose‐dependent manner. NK012 suppressed the progression of hind‐leg paralysis and prolonged the survival time of the mice compared to the untreated control group. In combination with bortezomib (BTZ), NK012 increased the median survival time compared to that with BTZ alone. In conclusion, these results suggest that NK012 is a potential candidate for use—alone and in combination—in the treatment of MM in humans.

An in vivo mechanism for the reduced peripheral neurotoxicity of NK105: a paclitaxel-incorporating polymeric micellar nanoparticle formulation

In our previous rodent studies, the paclitaxel (PTX)-incorporating polymeric micellar nanoparticle formulation NK105 had showed significantly stronger antitumor effects and reduced peripheral neurotoxicity than PTX dissolved in Cremophor® EL and ethanol (PTX/CRE). Thus, to elucidate the mechanisms underlying reduced peripheral neurotoxicity due to NK105, we performed pharmacokinetic analyses of NK105 and PTX/CRE in rats. Among neural tissues, the highest PTX concentrations were found in the dorsal root ganglion (DRG). Moreover, exposure of DRG to PTX (Cmax_PTX and AUC0-inf._PTX) in the NK105 group was almost half that in the PTX/CRE group, whereas exposure of sciatic and sural nerves was greater in the NK105 group than in the PTX/CRE group. In histopathological analyses, damage to DRG and both peripheral nerves was less in the NK105 group than in the PTX/CRE group. The consistency of these pharmacokinetic and histopathological data suggests that high levels of PTX in the DRG play an important role in the induction of peripheral neurotoxicity, and reduced distribution of PTX to the DRG of NK105-treated rats limits the ensuing peripheral neurotoxicity. In further analyses of PTX distribution to the DRG, Evans blue (Eb) was injected with BODIPY®-labeled NK105 into rats, and Eb fluorescence was observed only in the DRG. Following injection, most Eb dye bound to albumin particles of ~8 nm and had penetrated the DRG. In contrast, BODIPY®–NK105 particles of ~90 nm were not found in the DRG, suggesting differential penetration based on particle size. Because PTX also circulates as PTX–albumin particles of ~8 nm following injection of PTX/CRE, reduced peripheral neurotoxicity of NK105 may reflect exclusion from the DRG due to particle size, leading to reduced PTX levels in rat DRG (275).

Long-lasting immunosuppressive effects of tacrolimus-loaded micelle NK61060 in preclinical arthritis and colitis models

AIM Tacrolimus (TAC) is an important drug for inflammatory diseases. However, TAC has several limitations, such as variable trough concentrations among individuals and a high medication frequency. In this study, we created NK61060, a novel micellar TAC formulation, to circumvent these disadvantages. MATERIALS & METHODS Immunosuppressive activity of NK61060 was determined in the collagen-induced arthritis rat model, mannan-induced arthritis mouse model and dextran sodium sulfate-induced colitis mouse model. The pharmacokinetics and toxicology of NK61060 were evaluated in those models. RESULTS In arthritis and colitis models, NK61060 exhibited superior immunosuppressive activity compared with that of TAC. Pharmacokinetic and toxicological analyses indicated that NK61060 had a wider safety margin and could be administered at a reduced medication frequency. CONCLUSION NK61060 mitigates the trough concentration variability and the medication frequency and it may be a safer and more effective option for use in clinical settings. Further studies are needed to determine its clinical usefulness.

Abstract 4435: Anti-multiple myeloma activity of NK012, a micelle-forming macromolecular prodrug of SN-38, in an orthotopic model

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL NK012 is a micelle-forming macromolecular prodrug of SN-38, an active metabolite of irinotecan (CPT-11). It accumulates in tumor tissue and gradually releases SN-38 in an enzyme-independent manner. We previously reported that NK012 showed stronger antitumor activity in a broad range of human solid tumor xenograft models than CPT-11 (2006 AACR Annual Meeting, #3062). Currently, phase II clinical trials of NK012 in patients with triple-negative breast cancer, small cell lung cancer, and colorectal cancer are ongoing in the United States and Japan. In this study, we evaluated the antitumor activity of NK012 in an orthotopic multiple myeloma model that shows typical infiltration of human myeloma cells into murine bone marrow tissue. A suspension of 2 × 106 cells of CD138 positive U266 human myeloma cells was injected into immunodeficient NOG (NOD/Shi-scid, IL-2Rγnull) mice via tail vein. The transplanted mice produced plasma M protein (human IgE) and developed lytic bone lesions. In addition, hind-leg paralysis caused by myeloma progression in spinal cord was observed. These symptoms were similar to complications observed in the patients with multiple myeloma. The intravenous administration of NK012 at 3.75, 7.5, and 15 mg/kg/day by q4d × 3 suppressed M protein concentration in plasma and proliferation of myeloma cells in bone marrow in a dose-dependent manner. Also observed were decrease in the area of osteolytic lesions and recovery of decreased total bone mineral density in the tibias. To examine the efficacy of NK012 in survival time, the myeloma-transplanted mice were treated with 9.4 mg/kg/day (28.2 mg/m2/day), q7d × 6, equivalent to the clinical recommended dose of 28 mg/m2/day as a single dose. NK012 significantly prolonged survival time of the mice compared with control group (78.5 days vs. 40 days, P<0.001). We next examined the combination effect of NK012 with bortezomib, which is commonly used for multiple myeloma treatment. The combined treatment of NK012 (9.4 mg/kg/day, q7d × 3) with bortezomib (0.5 mg/kg/day, 2 times/week × 2, i.v.) exhibited increase in median survival time over bortezomib alone (83.5 days vs. 68 days). In conclusion, these results suggested NK012 is a promising candidate for human multiple myeloma treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4435. doi:10.1158/1538-7445.AM2011-4435

Abstract 5513: Synergistic antitumor activity of NK012, a micelle-forming macromolecular prodrug of SN-38, combined with platinum compounds in human cancer xenograft models

Irinotecan (CPT-11) is clinically used to treat various tumors, but low enzymatic conversion to its active metabolite, SN-38, limits its therapeutic effect. NK012, a micelle-forming macromolecular prodrug of SN-38, accumulates in tumor tissue and gradually releases SN-38 in enzyme-independent manner. NK012 showed much stronger growth inhibitory effects than CPT-11 at maximum tolerated dose (MTD) against all tested xenografted human tumors. Furthermore, NK012 showed much stronger antitumor activities than other cytotoxic agents clinically used for cancer treatment (taxanes, doxorubicin, carboplatin etc.). Here, we report combination antitumor effects of NK012 with platinum compounds in human breast cancer, non-small cell lung cancer, colorectal cancer and hepatocellular cancer xenografts in nude mice. The dose of NK012 was set at 1.9-3.8 mg/kg/day to be sub-optimum for complete regression depending on the cell line. NK012 and carboplatin were administered by q7dx3. Although both of NK012 and carboplatin were ineffective as single agent (minimum T/C values >50%), their combination was effective (minimum T/C value = 8%) to cause tumor regression in MC-05-JCK breast cancer. In MC-05-JCK breast cancer, the TGDs (Tumor Growth Delay; tumor tripling time over control group delayed by treatment / tumor tripling time of control group × 100) by NK012 1.9 mg/kg/day, carboplatin 50 mg/kg/day (67% of MTD), and their combination were 41%, 29%, and 286%, respectively. In NCI-H460 non-small cell lung cancer, the TGDs by NK012 3.8 mg/kg/day, carboplatin 75 mg/kg/day (MTD), and their combination were 90%, 71%, and 403%, respectively. In Co-3 colorectal cancer, the TGDs by NK012 3.8 mg/kg/day, carboplatin 75 mg/kg/day, and their combination were 358%, 97%, and >489%, respectively. Thus, combination of NK012 with carboplatin showed remarkably synergistic effects, and it didn9t accompany toxicity exacerbation. Similarly, combination of NK012 with cisplatin exhibited remarkable synergistic effects in Li-07-JCK hepatocellular cancer. The TGDs by NK012 3.8 mg/kg, one shot, cisplatin 10 mg/kg, one shot, and their combination were 58%, 136%, and 455%, respectively. These results indicate that NK012 and platinum compounds combination regimen would be effective for treatment of patients with solid tumors including breast, non-small cell lung, colorectal and hepatocellular carcinomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5513.

Abstract 5522: Comparison of pharmacokinetics/pharmacodynamics of NK012, a micelle-forming prodrug of SN-38, and CPT-11 in human breast cancer xenograft model

Introduction: NK012 is a novel micelle-forming macromolecular prodrug of SN-38, an active metabolite of irinotecan (CPT-11), which is gradually released from the molecule in an enzyme-independent manner at physiological pH. We have demonstrated that antitumor activity of NK012 at 3.8 mg/kg/day by q4d×3 (1/8 MTD) and above was superior to that of CPT-11 at its MTD (67 mg/kg by q4d×3) against human breast cancer MC-05-JCK, and a phase II clinical trial of NK012 is ongoing against triple-negative breast cancer in the US. In the present study, we compared pharmacokinetics and pharmacodynamics of NK012 with those of CPT-11 in human breast cancer MC-05-JCK-bearing nude mice. Methods: NK012 (3.8 and 30 mg/kg) and CPT-11 (67 mg/kg) were given intravenously to nude mice bearing MC-05-JCK, and microdialysis probe was placed in the tumor tissue to determine free SN-38 concentration in tumor extracellular fluid (ECF). The drug concentrations in the plasma and tumor tissue were determined by HPLC system. Intratumoral distribution of NK012 was also investigated by observing auto-fluorescence of the drug with a fluorescence microscope. Cytotoxic effects of the drugs were examined immunohistochemically using anti-phosph γH2AX and anti-phospho histone H3 antibodies to detect cleaved DNA and M-phase cells, respectively. Results: After a single NK012 administration, polymer-bound SN-38 and polymer-unbound SN-38 (released SN-38) showed remarkably prolonged blood circulation. On the other hand, CPT-11 and its active metabolite SN-38 were promptly removed from the circulation. NK012 also retained in the tumor tissue for a long period as compared with CPT-11. Microdialysate experiment revealed that the free SN-38 from NK012 in the tumor ECF was eliminated more slowly than that converted from CPT-11. NK012-originated fluorescence in the tumor tissue was mainly observed in the stroma, necrotic area and perivascular region. The phosphorylation of γH2AX due to DNA breakage and consequent reduction in M-phase cells were observed for a longer period in NK012-treated tumor tissue than that treated with CPT-11. HE staining of MC-05-JCK revealed that apoptotic tumor cells appeared 3 days after administration of both NK012 and CPT-11 injection, but fibrosis in the tumor stroma was only observed with NK012 treatment (day 10). These histopathological changes caused by the drugs were associated with the elimination rate of SN-38 in the tumor tissue. Conclusion: The superior antitumor efficacy of NK012 to CPT-11 was associated with the sustained release of free SN-38 from the NK012 accumulated in tumor tissue as well as the prolonged circulation of NK012 in the bloodstream. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5522.